1. Field of the Invention
This invention pertains to the field of compositions and methods that enhance the delivery of glucocorticoid and ascomycin drugs, such as hydrocortisone, cyclosporin and FK506, across the skin for the treatment of psoriasis and other inflammatory diseases of the skin.
2. Background
Transdermal or transmucosal drug delivery is an attractive route of drug delivery for several reasons. Gastrointestinal drug degradation and the hepatic first-pass effect are avoided. In addition, transdermal and transmucosal drug delivery is well-suited to controlled, sustained delivery (see, e.g., Elias, In Percutaneous Absorption: Mechanisms-Methodology-Drug Delivery, Bronaugh and Maibach, Eds., pp 1-12, Marcel Dekker, New York, 1989.). For many applications, traditional methods of administering drugs are not optimal because of the very large initial concentration of the drug. Transdermal delivery could allow a more uniform, slower rate of delivery of a drug. Moreover, patient compliance is encouraged because such delivery methods are easy to use, comfortable, convenient and non-invasive.
These advantages of transdermal and transmucosal delivery have not led to many clinical applications because of the low permeability of epithelial membranes, the skin in particular, to drugs. The difficulties in delivering drugs across the skin result from the barrier property of skin. Skin is a structurally complex thick membrane that represents the body""s border to the external hostile environment. The skin is composed of the epidermis, the dermis, the hypodermis, and the adenexal structures (epidermal appendages). The epidermis, the outermost epithelial tissue of the skin, is itself composed of several layersxe2x80x94the stratum corneum, the stratum granulosum, the stratum spinosum, and the stratum basale.
Compounds that move from the environment into and through intact skin must first penetrate the stratum corneum, the outermost layer of skin, which is compact and highly keratinized. The stratum corneum is composed of several layers of keratin-filled skin cells that are tightly bound together by a xe2x80x9cgluexe2x80x9d composed of cholesterol and fatty acids. The thickness of the stratum corneum varies depending upon body location. It is the presence of stratum corneum that results in the impermeability of the skin to pharmaceutical agents. The stratum corneum is formed naturally by cells migrating from the basal layer toward the skin surface where they are eventually sloughed off. As the cells progress toward the surface, they become progressively more dehydrated and keratinized. The penetration across the stratum corneum layer is generally the rate-limiting step of drug permeation across skin. See, e.g., Flynn, G. L., In Percutaneous Absorption: Mechanisms-Methodology-Drug Delivery, supra. at pages 27-53.
After penetration through the stratum corneum layer, systemically acting drug molecules then must pass into and through the epidermis, the dermis, and finally through the capillary walls of the bloodstream. The epidermis, which lies under the stratum corneum, is composed of three layers. The outermost of these layers is the stratum granulosum, which lies adjacent to the stratum corneum, is composed of cells that are differentiated from basal cells and keratinocytes, which make up the underlying layers having acquired additional keratin and a more flattened shape. The cells of this layer of the epidermis, which contain granules that are composed largely of the protein filaggrin. This protein is believed to bind to the keratin filaments to form the keratin complex. The cells also synthesize lipids that function as a xe2x80x9ccementxe2x80x9d to hold the cells together. The epidermis, in particular the stratum granulosum, contains enzymes such as aminopeptidases.
The next-outermost layer of the epidermis is the stratum spinosum, the principal cells of which are keratinocytes, which are derived from basal cells that comprise the basal cell layer. Langerhans cells, which are also found in the stratum spinosum, are antigen-presenting cells and thus are involved in the mounting of an immune response against antigens that pass into the skin. The cells of this layer are generally involved in contact sensitivity dermatitis.
The innermost epidermal layer is the stratum basale, or basal cell layer, which consists of one cell layer of cuboidal cells that are attached by hemi-desmosomes to a thin basement membrane which separates the basal cell layer from the underlying dermis. The cells of the basal layer are relatively undifferentiated, proliferating cells that serve as a progenitor of the outer layers of the epidermis. The basal cell layer includes, in addition to the basal cells, melanocytes.
The dermis is found under the epidermis, which is separated from the dermis by a basement membrane that consists of interlocking rete ridges and dermal papillae. The dermis itself is composed of two layers, the papillary dermis and the reticular dermis. The dermis consists of fibroblasts, histiocytes, endothelial cells, perivascular macrophages and dendritic cells, mast cells, smooth muscle cells, and cells of peripheral nerves and their end-organ receptors. The dermis also includes fibrous materials such as collagen and reticulin, as well as a ground substance (principally glycosaminoglycans, including hyaluronic acid, chondroitin sulfate, and dermatan sulfate).
Several methods have been proposed to enhance transdermal transport of drugs. For example, chemical enhancers (Burnette, R. R. In Developmental Issues and Research Initiatives; Hadgraft J., Ed., Marcel Dekker: 1989; pp. 247-288), iontophoresis, and others have been used. However, in spite of the more than thirty years of research that has gone into delivery of drugs across the skin in particular, fewer than a dozen drugs are now available for transdermal administration in, for example, skin patches.
Transport of drugs and other molecules across the blood-brain barrier is also problematic. The brain capillaries that make up the blood-brain barrier are composed of endothelial cells that form tight junctions between themselves (Goldstein et al., Scientific American 255:74-83 (1986); Pardridge, W. M., Endocrin. Rev. 7: 314-330 (1986)). The endothelial cells and the tight intercellular junctions that join the cells form a barrier against the passive movement of many molecules from the blood to the brain. The endothelial cells of the blood-brain barrier have few pinocytotic vesicles, which in other tissues can allow somewhat unselective transport across the capillary wall. Nor is the blood-brain barrier interrupted by continuous gaps or channels that run through the cells, thus allowing for unrestrained passage of drugs and other molecules.
Thus, a need exists for improved reagents and methods for enhancing delivery of compounds, including drugs, across epithelial tissues and endothelial tissues such as the skin and the blood-brain barrier. The present invention fulfills this and other needs.
The present invention provides methods for treating a skin inflammatory condition. In some embodiments, the methods comprise contacting skin affected by the inflammatory condition with a conjugate that comprises a) a glucocorticoid or an ascomycin, and b) a delivery-enhancing transporter that comprises 5 to 25 arginine residues. Examples of inflammatory conditions include psoriasis, eczema and alopecia areata. In some embodiments, the conjugate is applied to intact skin.
The invention also provides for conjugates that comprise a) a glucocorticoid or an ascomycin, and b) a delivery-enhancing transporter that comprises 5 to 25 arginine residues.
The glucocorticoid can be hydrocortisone, for example. Examples of ascomycins include cyclosporin and FK506. In some embodiments, the cyclosporin is cyclosporin A.
In some embodiments, the delivery-enhancing transporter comprises 7-15 arginine residues. The delivery-enhancing transporter can have at least one arginine that is a D-arginine and in some embodiments, all arginines are D-arginine.
The delivery-enhancing transporter can consist essentially of 5 to 50 amino acids, at least 50 percent of which are arginine. In some embodiments, at least 70% of the amino acids are arginines. In some embodiments, the delivery-enhancing transporter comprises at least 5 contiguous arginines.
The glucocorticoid or ascomycin can be connected to the delivery enhancing transporter by a linker. In some embodiments, the linker is a releasable linker which releases the glucocorticoid or ascomycin, in biologically active form, from the delivery-enhancing transporter after the glucocorticoid or ascomycin has passed into and through one or more layers of the skin. In some embodiments, the ascomycin or glucocorticoid is released from the linker by solvent-mediated cleavage. In some embodiments, the conjugate is substantially stable at acidic pH but the ascomycin or glucocorticoid is substantially released from the delivery-enhancing transporter at physiological pH. In some embodiments, the half-life of the conjugate is between 5 minutes and 24 hours upon contact with the skin. For example, the half-life can be between 30 minutes and 2 hours upon contact with the skin.
Examples of conjugate structures include the group consisting of structures 3, 4, or 5, as follows: 
where R1xe2x80x94X comprises the glucocorticoid or ascomycin; X is a functional group on the glucocorticoid or ascomycin to which the linker is attached; Y is N or C; R2 is hydrogen, alkyl, aryl, acyl, or allyl; R3 comprises the delivery-enhancing transporter; R4 is substituted or unsubstituted S, O, N or C; R5 is OH, SH or NHR6; R6 is hydrogen, alkyl, aryl, acyl or allyl; k and m are each independently selected from 1 and 2; and n is 1 to 10. Preferably, X is selected from the group consisting of N, O, S, and CR7R8, wherein R7 and R8 are each independently selected from the group consisting of H and alkyl. In some embodiments, R4 is S; R5 is NHR6; and R6 is hydrogen, methyl, allyl, butyl or phenyl. In some embodiments, R2 is benzyl; k, m, and n are each 1, and X is O. In some embodiments, the conjugate comprises structure 3 and R2 is selected to obtain a conjugate half-life of between 5 minutes and 24 hours. In some embodiments, R2 is selected to obtain a conjugate half-life of between 5 minutes and 24 hours. In some embodiments, the conjugate comprises structure 4; R4 is S; R5 is NHR6; and and is hydrogen, methyl, allyl, butyl or phenyl. In some embodiments, the conjugate comprises structure 4; R5 is NHR6; R6 is hydrogen, methyl, allyl, butyl or phenyl; and k and m are each 1. One example of a conjugate is: 
where Ph is phenyl.
The invention also provides conjugates in which the release of the linker from the biological agent involves a first, rate-limiting intramolecular reaction, followed by a faster intramolecular reaction that results in release of the linker. The rate-limiting reaction can, by appropriate choice of substituents of the linker, be made to be stable at a pH that is higher or lower than physiological pH. However, once the conjugate has passed into and across one or more layers of an epithelial or endothelial tissue, the linker will be cleaved from the agent. An example of this type of linker is structure 6, as follows: 
wherein R1xe2x80x94X comprises the glucocorticoid or ascomycin; X is a functional group on the glucocorticoid or ascomycin to which the linker is attached; Ar is an aryl group having the attached radicals arranged in an ortho or para configuration, which aryl group can be substituted or unsubstituted; R3 comprises the delivery-enhancing transporter; R4 is substituted or unsubstituted S, O, N or C; R5 is OH, SH or NHR6; R6 is hydrogen, alkyl, aryl, acyl or allyl; and k and m are each independently selected from 1 and 2. In some embodiments, X is selected from the group consisting of N, O, S, and CR7R8, wherein R7 and R8 are each independently selected from the group consisting of H and alkyl. In some embodiments, R4 is S; R5 is NHR6; and R6 is hydrogen, methyl, allyl, butyl or phenyl. In some embodiments, the conjugate comprises: 
In preferred embodiments, the compositions of the invention comprise a linker susceptible to solvent-mediated cleavage. For example, a preferred linker is substantially stable at acidic pH but is substantially cleaved at physiological pH.